Journal of Biological Chemistry
Volume 277, Issue 40, 4 October 2002, Pages 37369-37376
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MOLECULAR BASIS OF CELL AND DEVELOPMENTAL BIOLOGY
A Vacuolar-type H+-Pyrophosphatase Governs Maintenance of Functional Acidocalcisomes and Growth of the Insect and Mammalian Forms of Trypanosoma brucei*

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Vacuolar proton pyrophosphatases (V-H+-PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H+-PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H+-PPase gene by using double-stranded RNA interference methodology to produce inducible V-H+-PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1). Acidocalcisomes of these mutated parasites lost acidity and contained 90% less polyphosphate. PFiVP1 did not release calcium after the addition of nigericin, and its total acidity was reduced by 70%. This mutant also failed to stabilize its intracellular pH on exposure to external basic pH >7.4 and recovered from intracellular acidification at a slower rate and to a more acidic final intracellular pH. In the absence of T. bruceiV-H+-PPase expression, PFiVP1 and BFiVP1 grew at a slower rate with doubling times of 27 h instead of 15 h, and 10 h instead of 7.5 h, respectively. Moreover, BFiVP1 could not grow over 5 × 105 cells/ml corresponding to a cell density reduction of five times for bloodstream form stationary phase growth.

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Published, JBC Papers in Press, July 16, 2002, DOI 10.1074/jbc.M204744200

*

This work was supported by the CNRS, the Conseil Régional d'Aquitaine, the GDR CNRS-Parasitologie and the Ministère de l'Education Nationale de la Recherche et de la Technologie (Action Microbiologie), and the United Nations Development Project/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (to R. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s)AF159881 and AY043295.